The main objective is to study the sequence of events that lead progressively to frank neoplasia. To do this effectively, it is necessary to be able to separate out discrete cell populations identified as particular stages in this sequence of events and to follow the progression of neoplasia in the cell populations evolving from the original cells. An in vivo-in vitro approach is used to identify, in a nondestructive manner, the lesions on pieces of DMBA-preexposed rat tracheal implants from the cytopathology in the cells which exfoliate from these pieces in organ culture. Cell populations are then generated directly from the pieces as outgrowths of epithelial cells in primary culture, and markers of the progression of neoplasia are looked for in these cell cultures and the cell lines established from them. The plan is to use this approach to investigate the cellular and biochemical properties of the early to late events in the progression of neoplasia in respiratory epithelium and to correlate morphological markers with these properties. To carry this out, the first aim is to quantitate the effects of DMBA exposure conditions on the numbers of carcinogen-altered cell populations induced, and the persistence of these populations after completion of exposure. These carcinogen-altered cell populations are separated out from normal cells by the selective survival of the altered cells in pyruvate and insulin-deprived medium. The second aim is to investigate the underlying mechanisms which permit survival of carcinogen-altered cells in the pyruvate and insulin-deprived (selection) medium, but allow for long-term growth of normal tracheal cells in the presence of these metabolites. To do this, the differences in utilization of key metabolites for biosynthetic pathways and energy production will be studied in the normal and carcinogen-altered cell populations. Once some patterns begin to emerge, changes will be looked for in the activity of a key enzyme(s) which may be associated with the regulation of this enzyme(s) during carcinogenesis in the future. The third aim is to continue studies of the effects of the promoter TPA on the early stages, i.e. the numbers of carcinogen-altered cell populations induced in the tracheal implants, as well as, the enhancement of late stages, i.e. increase in the expression of severe atypias in vivo and other late markers (multinucleation in the presence of cytochalasin B, tumor formation) of neoplasia in vitro.